A conserved biological response to double-stranded RNA (dsRNA) mediates resistance to parasitic nucleic acids, encoded by transposable elements and viruses. Cleavage of dsRNA into small interfering RNAs (siRNAs) triggers repression of genes related by sequence to the siRNAs. A large number of siRNAs made by cells correspond to endogenous mRNAs, and these endogenous siRNAs (endo-siRNAs) trigger modest repression on their targets. This silencing mechanism is related to another endogenous regulatory process that represses the expression of most protein-coding genes in the genome. The latter process is guided by microRNAs (miRNAs), which are produced from hundreds of genes within the genome. The long-term goals of our research are to understand the biological functions of endo-siRNAs and miRNAs, and to understand what aspects of cell behavior are controlled by small non-coding RNA regulation. To this end, we have developed genetic, biochemical and cell biological methods in whole Drosophila and cells to achieve our goals. In previous work, we conducted molecular genetic experiments to discover regulatory functions of endo-siRNAs and miRNAs. Results of those projects are the basis for research aims in this proposal. Our aims are to: 1) determine how endo-siRNAs make early embryonic development resistant to environmental variation mediated by temperature, 2) determine whether miRNAs suppress noise in gene expression by creating thresholds of protein production, 3) understand how energy metabolism controls miRNA activity and why miRNAs become dispensable to cells under metabolically restricted conditions. The significance to health is manifold. The ability of small RNAs to regulate organismal gene expression suggests that basic understanding will reveal control mechanisms that influence many aspects of biology. It will have an impact on non-infectious diseases such as cancer, where miRNA genes are frequently mutated. This work shows that miRNAs can suppress the effects of genome variation between individuals on creating variation in critical cell behaviors. In the coming personal genome era of medicine, understanding the extent of genome suppression will be critical for disease prediction and prevention methods that rely on genome sequence. Finally, small RNAs are used diagnostically for disease prognosis, and are being commercially developed as disease therapeutics, making basic understanding of these molecules an important biomedical research goal.